Treatment of hyperandrogenic conditions

ABSTRACT

The invention concerns the treatment of hyperandrogenic conditions in humans by the formation of a novel mechanism-based irreversible inhibitor of human 5α-reductase enzymes from 3-oxo-4-oxa end 4-thiasteroids having a 1,2-double bond and the pyridine-nucleotide cofactor of the 5α-reductase enzyme, NADPH. The invention further relates to the isolated inhibitor-cofactor complex.

CROSS-REFERENCE

This application claims the benefit of Provisional Application No.60/005,869 filed Oct. 26. 1965.

SUMMARY OF THE INVENTION

The invention concerns the treatment of hyperandrogenic conditions inhumans by the formation of a novel mechanism-based irreversibleinhibitor of human 5α-reductase enzymes from 3-oxo-4-oxa and4-thiasteroids having a 1,2-double bond and the pyridine-nucleotidecofactor of the 5α-reductase enzyme, NADPH. The invention furtherrelates to the isolated inhibitor-cofactor complex.

BACKGROUND OF THE INVENTION

Certain undesirable physiological manifestations, such as acne vulgaris,seborrhea, female hirsutism, androgenic alopecia which includes femaleand male pattern baldness, and benign prostatic hyperplasia, are theresult of hyperandrogenic stimulation caused by excessive accumulationof testosterone ("T") or similar androgenic hormones in the metabolicsystem. Androgenic alopecia is also known as androgenetic alopecia.Early attempts to provide a chemotherapeutic agent to counter theundesirable results of hyperandrogenicity resulted in the discovery ofseveral steroidal antiandrogens having undesirable hormonal activitiesof their own. The estrogens, for example, not only counteract the effectof the androgens but have a feminizing effect as well. Non-steroidalantiandrogens have also been developed, for example,4'-nitro-3'-trifluoromethyl-isobutyranilide. See Neri, et al.,Endocrinol. 1972, 91 (2). However, these products, though devoid ofhormonal effects, compete with all natural androgens for receptor sites,and hence have a tendency to feminize a male host or the male fetus of afemale host and/or initiate feed-back effects which would causehyperstimulation of the testes.

The principal mediator of androgenic activity in some target organs,e.g. the prostate, is 5α-dihydrotestosterone ("DHT"), formed locally inthe target organ by the action of testosterone-5α-reductase. Inhibitorsof testosterone-5α-reductase will serve to prevent or lessen symptoms ofhyperandrogenic stimulation in these organs.

The enzyme 5α-reductase catalyzes the reduction of testosterone to themore potent androgen, dihydrotestosterone, as shown below: ##STR1##

Finasteride,(17β-(N-tert-butylcarbamoyl)-3-oxo-4-aza-5α-androst-1-ene-3-one) asshown below, is a potent inhibitor of the human prostate enzyme.##STR2## Under the trade name PROSCAR®, finasteride is known to beuseful in the treatment of hyperandrogenic conditions; see eg. U.S. Pat.No. 4,760,071. Finasteride is currently prescribed for the treatment ofbenign prostatic hyperplasia (BPH), a condition afflicting to somedegree the majority of men over age 55. Finasteride's utility in thetreatment of androgenic alopecia and prostatic carcinoma is alsodisclosed in the following documents: EP 0 285,382, published 5 Oct.1988; EP 0 285,383, published 5 Oct. 1988; Canadian Patent no.1,302,277; and Canadian Patent no. 1,302,276.

There are two isozymes of 5α-reductase in humans. One isozyme (type 1)predominates in sebaceous glands of most regions of skin tissue and isrelatively insensitive to finasteride; the other (type 2) predominatesin the prostate and is potently inhibited by finasteride.

In clinical trials, the efficacy of finasteride far exceededexpectations based on its perceived potency against the human prostateenzyme, for which finasteride was first thought to be a simple,rapidly-reversible inhibitor with K_(i) =26 nM. For instance,circulating concentrations of finasteride comparable to this Ki actuallyreduced levels of dihydrotestosterone to values approaching those foundin individuals genetically deficient in the prostate isozyme, and aslong as two weeks were required for dihydrotestosterone to return tobasal levels after withdrawal of finasteride (Stoner, J. Steroid.Biochem. Molec. Biol. 37: 375-378 (1990) and Gormley et al., J. Clin.Endocrinol. Metabol. 70: 1136-1141 (1990)). A closer evaluation of theinteraction of finasteride with the human prostate (type 2) isozyme ledto appreciation that finasteride and certain analogs thereof areslow-binding inhibitors, such that their potency had been mistakenlyunderrated in standard fixed-time assays (Harris et al., Proc. Natl.Acad. Sci. U.S.A., 89: 10787-10791 (1992)). Independently, Faller etal., also recognized the inconsistency. Faller et al., have recentlydescribed in detail the slow-binding behavior of finasteride, reachingthe conclusion that finasteride binds to the human prostate isozyme witha rate constant of 2.7×10⁵ M⁻¹ s⁻¹ to form an essentially irreversibleenzyme-inhibitor complex with a K_(i) <<1 nM (Faller et al.,Biochemistry 32: 5705-5710 (1993)).

Although finasteride is not a significant inhibitor of human skin(type 1) isozyme at doses employed in the treatment of BPH, finasteridedoes slowly form a comparable high affinity complex with this isozyme.As determined by Tian, et al., Biochemistry 33: 2291-2296 (1994), thesecond-order rate constant for formation of this complex is 4.0×10³ M⁻¹s⁻¹, which is about 1% of the rate constant against the prostateisozyme. Based on the apparent irreversible inhibition and onstructure-activity considerations, Tian et al., proposed thatfinasteride binds to the enzyme covalently as a Michael acceptor. U.S.Ser. No. (08/368,513), filed Jan. 4, 1995, describes that finasterideand other 3-oxo-4-aza steroids having a 1,2 double bond aremechanism-based irreversible inhibitors of 5α-reductase.

The present invention demonstrates, in contrast to the expectation inthe art, that 3-oxo-4-oxa and 4-thiasteroids having a 1,2 double bondare novel mechanism-based irreversible inhibitors of 5α-reductase. These3-oxo-4-oxa and 4-thiasteroids having a 1,2-double bond are recognizedas a substrate by the human 5α-reductase type 1 and type 2 enzymes, andin the course of the enzymatic reduction, the 3-oxo-4-oxa and4-thiasteroid having a 1,2-double bond forms a covalent adduct with thepyridine-nucleotide cofactor (NADPH). The covalent inhibitor-cofactorcomplex formed between the 3-oxo-4-oxasteroid anion and the oxidizednicotinamide cofactor is bound by the enzyme as a potentcollected-product inhibitor.

Kaplan et al. averse et al., Bioorganic Chem. 1: 207-233 (1971)) intheir work with pyridine nucleotide-linked dehydrogenases identifiedanalogous high-affinity, abortive ternary complexes formed spontaneously(in the reverse direction) by several pyridine nucleotide-linkeddehydrogenases, notably lactate dehydrogenase. Lactate dehydrogenasemistakenly adds pyruvate to NAD⁺ to form the inhibitor shown below:##STR3## wherein B⁻ represents a basic group on the enzyme active siteand ADPR represents adenosine-diphospho-ribose.

DETAILED DESCRIPTION OF THE INVENTION

Novel mechanism-based irreversible inhibitors of human 5α-reductaseenzymes are formed from 3-oxo-4-oxa and 4-thiasteroids having a1,2-double bond and the pyridine-nucleotide cofactor of the 5α-reductaseenzyme NADPH. The structure of the covalent NADP-inhibitor complex, isrepresented below as structural formula (I): ##STR4## wherein X isoxygen or sulfur and (P)ADPR signifies2-phosphoadenosine-diphospho-ribose.

The covalent adduct of structural formula (I) may optionally be modifiedas known to those in the art so long as the 3-oxo position and theoxygen or sulfur at the 4-position are conserved. Particularly preferredare compounds substituted at any of the following positions of theoxasteroid ring: 7,15,16 and 17, depending on the structure of the3-oxo-4-oxa and 4-thiasteroid having a 1,2-double bond administered tothe human being.

The mechanism for inhibition of the 5α-reductase enzyme is proposed inScheme 1. There is a close parallel between reduction of the naturalsubstrate, testosterone, and reduction of 3-oxo-4-oxa and 4-thiasteroidinhibitors having a 1,2-double bond, since both proceed through nearlyidentical high-energy transition states. In the case of testosterone,transfer of hydride ion to C-5 gives the 3,4-enolate, and the reductionis completed by transfer of a proton to C-4 to allow tautomerization todihydrotestosterone. In the case of 3-oxo-4-oxa and 4-thiasteroidinhibitors having 1,2-double bond, transfer of hydride ion to C-1 givesthe 2,3-enolate instead. It is certain that this hydride transfer isessentially complete in the high-affinity complex, since nountransformed 3-oxo-4-oxa and 4-thiasteroid inhibitor having a1,2-double bond is recovered upon denaturation of the enzyme-inhibitorcomplex. At this point, the enzyme becomes trapped, being unprepared totransfer a proton to C-2 to complete the reduction. Unable to find aproton, the enolate/carbanion attacks the positively charged pyridiniumion of NADP⁺ to produce the covalent adduct, which the enzyme bindstenaciously. ##STR5## In the scheme above, AH and BH represent protondonors in the enzyme active site and (P)ADPR represents2-phospho-adenosine-diphospho-ribose.

3-oxo-4-oxa and 4-thiasteroids having a 1,2-double bond (also called Δ¹-3-oxo-4-oxa and 4-thiasteroids and Δ¹ -3-oxo-4-thiasteroids) areselectively activated by the enzyme 5α-reductase to produce the novelcovalent adduct with the cofactor NADPH of structural formula (I):##STR6## wherein X is oxygen or sulfur. Illustrating the 3-oxo-4-oxa and4-thiasteroids having a 1,2-double bond useful in the present inventionare the compounds of structural formula II, below: ##STR7## wherein X isoxygen or sulfur.

More particularly defining the 3-oxo-4-oxa and 4-thiasteroids having a1,2-double bond useful in the present invention are the compounds ofstructural formula III, below: ##STR8## or a pharmaceutically acceptablesalt, ester or stereoisomer thereof, wherein:

the C5-C6 bond designated with a dotted line independently represents asingle or double bond, provided that when the C5-C6 is a double bond,H_(a) is absent and when the C5-C6 bond is a single bond H_(a) ispresent and represents hydrogen;

X is selected from oxygen and sulfur;

R¹ is selected from hydrogen and C₁₋₅ alkyl;

R² is selected from CH₃, CH₂ OR³, and H;

R³ is selected from: C₁₋₅ alkyl;

Z is ##STR9## A¹ is selected from: (1) --H,

(2) keto,

(6) carboxy,

(7) protected amino,

(8) amino,

(9) C₁₋₁₀ alkyl,

(10) substituted or unsubstituted C₂₋₁₀ alkenyl,

(11) aryl-substituted C₁₋₁₀ alkyl,

(12) aryl or heteroaryl,

(13) substituted aryl or heteroaryl,

(14) aryl or heteroaryl carbamoyl-substituted C₁₋₁₀ alkyl,

(15) C₁₋₁₀ alkylcarbonyl,

(16) aryl or heteroaryl carbonyl,

(17) ether-substituted C₁₋₁₀ alkyl,

(18) thioether-substituted C₁₋₁₀ alkyl,

(19) keto-substituted C₁₋₁₀ alkyl,

(20) heteroaryl-substituted C₁₋₁₀ alkyl,

(21) carboxylic ester,

(22) carboxamide, including substituted and unsubstituted anilidederivatives,

(23) urea,

(24) C₁₋₁₀ alkylureido C₀₋₅ alkyl,

(25) substituted or unsubstituted heteroaryl or arylureidoC₀₋₅ alkyl,

(26) C₁₋₁₀ alkanoyloxyC₁₋₂ alkyl,

(27) C₁₋₁₀ alkylcarbonylamino,

(28) alkanoylamidoalkyl

(29) ether,

(30) thio ether, and

(31) substituted and unsubstituted aryl or heteroaryl ether;

A² is selected from:

(1) --H,

(2) keto,

(3) protected hydroxy,

(4) acetate,

(5) hydroxy,

(6) carboxy,

(7) protected amino,

(8) amino,

(9) C₁₋₁₀ alkyl,

(10) substituted or unsubstituted C₂₋₁₀ alkenyl,

(11) aryl-substituted C₁₋₁₀ alkyl,

(12) aryl or heteroaryl,

(13) substituted aryl or heteroaryl,

(14) aryl or heteroaryl carbamoyl-substituted C₁₋₁₀ alkyl,

(15) C₁₋₁₀ alkylcarbonyl,

(16) aryl or heteroaryl carbonyl,

(17) ether-substituted C₁₋₁₀ alkyl,

(18) thioether-substituted C₁₋₁₀ alkyl,

(19) keto-substituted C₁₋₁₀ alkyl,

(20) heteroaryl-substituted C₁₋₁₀ alkyl,

(21) carboxylic ester,

(22) carboxamide, including substituted and unsubstituted anilidederivatives,

(23) urea,

(24) C₁₋₁₀ alkylureido C₀₋₅ alkyl,

(25) substituted or unsubstituted arylureidoC₀₋₅ alkyl,

(26) C₁₋₁₀ alkanoyloxyC₁₋₂ alkyl,

(27) C₁₋₁₀ alkylcarbonylamino,

(28) alkanoylamidoalkyl,

(29) ether,

(30) thio ether, and

(31) substituted and unsubstituted aryl- or heteroaryl-ether;

Heteroaryl is selected from piperidinyl, piperizinyl, pyrrolidinyl,pyrrolyl, furanyl, thienyl, pyridyl, pyrimidinyl, indolyl andbenzofuranyl.

Preferred are compounds wherein:

(a) protected hydroxy is selected from: dimethyl-t-butyl silyloxy,trimethylsilyloxy, tri-ethylsilyloxy, tri-isopropylsilyloxy, andtriphenylsilyloxy;

(b) protected amino is acetylamino, benzoylamino, and pivaloylamino;

(c) C₁₋₁₀ alkyl is selected from methyl, ethyl, propyl, butyl, pentyl,1,5-dimethylhexyl, 6-methylhept-2-yl, 5-methylhexyl, and1-methyl-4-isopropylhexyl;

(d) substituted or unsubstituted C₂₋₁₀ alkenyl is selected from:phenylmethylene, chlorophenylmethylene, ethoxycarbonylphenylmethylene,carboxyphenylmethylene,(((1,1-dimethylethyl)amino)carbonyl)phenylmethylene, trimethoxyphenylmethylene, methoxyphenylmethylene, methylsulfonylphenylmethylene,biphenylmethylene, nitrophenylmethylene, aminophenylmethylene,acetylaminophenylmethylene, pivaloylaminophenylmethylene,phenoxyphenylmethylene, 2-imidazolyl methylene, 2-thiazolylmethylene,

(e) aryl substituted C₁₋₁₀ alkyl is selected from omega-phenylpropyl and1-(chlorophenoxy)ethyl;

(f) aryl is selected from phenyl, and naphthyl;

(g) substituted aryl or heteroaryl is selected from phenyl, pyridyl andpyrimidinyl substituted with one to three substituents independentlyselected from:

(1) --H,

(2) --OH,

(3) --CH₃,

(4) --OCH₃,

(5) --S(O)_(n) --CH₃, wherein n is selected from 0, 1, and 2,

(6) --CF₃,

(7) halo,

(8) --CHO,

(9) CN,

(10) phenyloxy,

(11) ethyl,

(12) t-butyl,

(13) OCH₂ CH₃,

(14) OC(CH₃)₃, and

(15) --NHR₇, wherein R⁷ is selected from: --H, --C₁₋₈ alkyl, --C₁₋₆alkylcarbonyl, --C₁₋₆ alkylsulfonyl, and --C₁₋₆ alkoxycarbonyl,

(h) aryl or heteroaryl carbamoyl substituted C₁₋₁₀ alkyl is selectedfrom 2-(4-pyridyl-carbamoyl)ethyl and 2-phenyl-ethyl;

(i) C₁₋₁₀ alkylcarbonyl is selected from isobutylcarbonyl andisopropylcarbonyl;

(j) aryl or heteroaryl carbonyl is selected from phenylcarbonyl andpyridyl carbonyl;

(k) ether-substituted C₁₋₁₀ alkyl is selected from 1-methoxy-ethyl and1-ethoxy-ethyl;

(l) thioether-substituted C₁₋₁₀ alkyl is selected from1-methylthio-ethyl, and 1-ethylthio-ethyl;

(m) keto-substituted C₁₋₁₀ alkyl is 1-keto-ethyl, ketomethyl,1-ketopropyl, and ketobutyl;

(n) heteroaryl-substituted C₁₋₁₀ alkyl is omega-(4-pyridyl)-butyl;

(o) carboxylic esters are C₁₋₁₀ alkylcarboxylic esters selected fromcarbomethoxy and carboethoxy;

(p) carboxamides are selected from N,N-diisopropyl carboxamide,N-t-butyl carboxamide, N-t-octyl carboxamide, N-n-octyl carboxamide,N-(hydroxyphenyl) carboxamide, N-phenylcarboxamide, N-(aminophenyl)carboxamide, N-(carbomethoxy)phenyl carboxamide, N-(methoxycarboxy)phenyl carboxamide, N-acetamidophenyl-N-acetyl-carboxamide,N-acetamidophenyl-carboxamide, N-pivalamidophenyl carboxamide,N-isobutyramidophenyl carboxamide, N-(methyl),N-diphenylmethyl)carboxamide, N-(diphenylmethyl)-carboxamide, N-t-butyl carboxamide,N-isopropyl carboxamide, 1-adamantyl carboxamide, 2-adamantylcarboxamide and N-(substituted phenyl) carboxamides wherein the phenylmay be substituted with 1 to 2 substitutents selected from ethyl,methyl, trifluoromethyl or halo (F, Cl, Br, I);

(q) C₁₋₁₀ alkanoyloxyC₁₋₂ alkyl is selected from acetyloxymethyl,trimethylacetyloxymethyl, and (2-ethylhexanoyloxy)methyl;

(r) urea is t-butylcarbonylamino urea;

(s) C₁₋₁₀ alkylureido C₀₋₅ alkyl is selected from:N-t-butylureidomethyl, N-n-propylureidomethyl, N-n-octylureidomethyl,N-isopropylureido, allylureido,

(t) substituted or unsubstituted arylureidoC₀₋₅ alkyl is selected from:N-(ethylphenyl) ureidomethyl, N-(chlorophenyl) ureidomethyl,N-phenylureidomethyl, N-(dichlorophenyl) ureidomethyl,N-naphth-2-yl)ureidomethyl, N-thiazol-2-ylureidomethyl,N-thien-2-ylmethylureidomethyl, N-(fluorophenyl)ureido,N-(methoxyphenyl)ureido, and 2-(ethoxyphenyl)ureidomethyl;

(u) C₁₋₁₀ alkylcarbonylamino is t-butylcarbonylamino;

(v) alkanoylamidoalkyl is selected from: trimethylacetamidomethyl,carbomethoxyoctanoylamidomethyl, (isobutylphenyl)propionamidomethyl,8-carboxyoctanoylamidomethyl, bromohexanoylamido methyl,hydroxydodecanoyl amidomethyl, 4-nitrophenylprionamidomethyl,isopropylthioacetamidomethyl, benzyloxyacetamidomethyl,carbomethoxyacetamidomethyl, triphenylproprionamidomethyl,cyclohexylacetamidomethyl, methylcyclohexanecarboxamidomethyl,(3-hydroxy-4,4,4-trichlorobutyramido)methyl, andphenylthio-acetamidomethyl;

(w) ether is selected from ethylene ketal, and C₁₋₈ alkyl etheroptionally substituted with hydroxy, halo, C₁₋₈ alkoxy, C₂₋₆ alkenyl, oraryl;

(x) thioether is selected from: C₁₋₈ alkylthio, phenylthio, and C₁₋₈alkylthio substituted with phenyl; and

(y) substituted and unsubstituted aryl or heteroaryl ether is selectedfrom thiophenoxy, biphenyloxy, acetamidophenoxy, (3-pyridyl)oxy,chlorophenyloxy, methylphenyloxy, phenoxy, hydroxyphenyloxy,methylsulfonylphenyloxy and pyrimidinyloxy.

In one embodiment of the instant invention are compounds of formula IIIwherein X is oxygen.

In one class of the compounds of this embodiment are compounds wherein Zis ##STR10##

Exemplifying this class are:

(1) N-t-Butyl-4-oxa-5α-androst-1-en-3-one-17β-carboxamide,

(2) 7β-Methyl-4-oxa-5α-cholest-1-en-3-one,

(3)17β-(2,5-bis(trifluoromethyl)phenylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(4)17β-(2,3-bis(trifluoromethyl)phenylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(5)17β-(2,4-bis(trifluoromethyl)phenylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(6)17β-(2,6-bis(trifluoromethyl)phenylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(7) 17β-(N-tert-amylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(8) 17β-(N-tert-hexylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(9) 17β-(N-isobutylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(10) 17β-(N-tert-octylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(11) 17β-(N-1,1-diethylbutylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(12) 17β-(N-neopentylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(13) 17β-(N-2-adamantylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(14) 17β-(N-1-adamantylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(15) 17β-(N-2-norbomylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(16) 17β-(N-1-norbomylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(17) 17β-(N-phenylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(18) 17β-(N-benzylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(19)17β-(2,3-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-oxa-5.alpha.-androst-1-en-3-one,

(20)17β-(2,4-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-oxa-5.alpha.-androst-1-en-3-one,

(21)17β-(2,6-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-oxa-5.alpha.-androst-1-en-3-one,

(22) 17β-(N-n-octylcarbamoyl)-4-methyl-4-oxa-5α-androst-1-en-3-one,

(23) 17β-(1-methoxyethyl)-7β-methyl-4-oxa-5α-androst-1-en-3-one

(24) 17β-(isopropyl)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(25) 17β-(4-methyl-phenoxy)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(26)17β-(1-(3-chlorophenoxy)ethyl)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(27)17β-(4-methylsulfonyl-phenoxy)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(28) 17β-(4-chlorophenoxy)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(29) 17β-(2-pyrimidinyloxy)-7β-methyl-4-oxa-5α-androst-1-en-3-one, and

(30)17β-(2,5-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-oxa-5.alpha.-androst-1-en-3-one.

In one subclass of this class are compounds wherein R¹ is hydrogen, R²is selected from H and CH₃, and A¹ is selected from: carboxamide,including substituted and unsubstituted anilide derivatives.

Further illustrating this subclass are compounds wherein carboxamide isselected from: N,N-diisopropyl carboxamide, N-t-butyl carboxamide,N-t-octyl carboxamide, N-n-octyl carboxamide, N-(hydroxyphenyl)carboxamide, N-phenylcarboxamide, N-(aminophenyl) carboxamide,N-(carbomethoxy)phenyl carboxamide, N-(methoxycarboxy) phenylcarboxamide, N-acetamidophenyl-N-acetyl-carboxamide,N-acetamidophenyl-carboxamide, N-pivalamidophenyl carboxamide,N-isobutyramidophenyl carboxamide, N-(methyl),N-(diphenylmethyl)carboxamide, N-(diphenylmethyl)-carboxamide, N-t-butyl carboxamide,N-isopropyl carboxamide, 1-adamantyl carboxamide, 2-adamantylcarboxamide, and N-(substituted phenyl) carboxamides wherein the phenylmay be substituted with 1 to 2 substitutents selected from ethyl,methyl, trifluoromethyl or halo (F, Cl, Br, I).

Still further illustrating this subclass are compounds whereincarboxamide is --C(═O)NH--C(CH₃)₃, --C(═O)NH--C₆ H₅ or--C(═O)NH--(2,5-trifluoromethylphenyl).

In one subclass of this class are compounds wherein R¹ is hydrogen, R²is selected from H and CH₃, the C5-C6 bond designated with a dotted lineis a single bond, H_(a) is present and represents hydrogen, and A¹ isselected from: carboxamide, including substituted and unsubstitutedanilide derivatives.

Further illustrating this subclass are compounds wherein carboxamide isselected from: N,N-diisopropyl carboxamide, N-t-butyl carboxamide,N-t-octyl carboxamide, N-n-octyl carboxamide, N-(hydroxyphenyl)carboxamide, N-phenylcarboxamide, N-(aminophenyl) carboxamide,N-(carbomethoxy)phenyl carboxamide, N-(methoxycarboxy) phenylcarboxamide, N-acetamidophenyl-N-acetyl-carboxamide,N-acetamidophenyl-carboxamide, N-pivalamidophenyl carboxamide,N-isobutyramidophenyl carboxamide, N-(methyl),N-(diphenylmethyl)carboxamide, N-(diphenylmethyl)-carboxamide, N-t-butyl carboxamide,N-isopropyl carboxamide, 1-adamantyl carboxamide, 2-adamantylcarboxamide, and N-(substituted phenyl) carboxamides wherein the phenylmay be substituted with 1 to 2 substitutents selected from ethyl,methyl, trifluoromethyl or halo (F, Cl, Br, I).

In one subclass of this class are compounds wherein R¹ is hydrogen, R²is selected from H and CH₃, the C5-C6 bond designated with a dotted lineis a double bond, H_(a) is absent, and Al is selected from: carboxamide,including substituted and unsubstituted anilide derivatives.

Further illustrating this subclass are compounds wherein carboxamide isselected from: N,N-diisopropyl carboxamide, N-t-butyl carboxamide,N-t-octyl carboxamide, N-n-octyl carboxamide, N-(hydroxyphenyl)carboxamide, N-phenylcarboxamide, N-(aminophenyl) carboxamide,N-(carbomethoxy)phenyl carboxamide, N-(methoxycarboxy) phenylcarboxamide, N-acetamidophenyl-N-acetyl-carboxamide,N-acetamidophenyl-carboxamide, N-pivalamidophenyl carboxamide,N-isobutyramidophenyl carboxamide, N-(methyl),N-(diphenylmethyl)carboxamide, N-(diphenylmethyl)-carboxamide, N-t-butyl carboxamide,N-isopropyl carboxamide, 1-adamantyl carboxamide, 2-adamantylcarboxamide and N-(substituted phenyl) carboxamides, wherein the phenylmay be substituted with 1 to 2 substitutents selected from ethyl,methyl, trifluoromethyl or halo (F, Cl, Br, I).

In another subclass of this class are compounds wherein R¹ is CH₃, R² isselected from H and CH₃, and A¹ is a selected from: carboxamide,including substituted and unsubstituted anilide derivatives, and C₁₋₁₀alkyl. Further illustrating this subclass are compounds whereincarboxamide is --C(═O)NH--C₆ H₅ or --C(═O)NH-(2,5-trifluoromethylphenyl)and C₁₋₁₀ alkyl is selected from isopropyl, isobutyl, 1,5-dimethylhexyl,and 5-methylhexyl.

In another class of the compounds of this embodiment are compoundswherein Z is ##STR11##

Exemplifying compounds of this class are:

(1) N-t-Butyl-4-oxa-5α-androst-1-en-3-one-16β-carboxamide,

(2) 16β-(1,5-dimethylhexyl)-7β-Methyl-4-oxa-5α-androst-1-en-3-one,

(3)16β-(2,5-bis(trifluoromethyl)phenylcarbamoyl)-4-oxa-androst-1-en-3-one,

(4) 16β-(N-tert-amylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(5) 16β-(N-tert-hexylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(6) 16β-(N-isobutylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(7) 16β-(N-tert-octylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(8) 16β-(N-1,1-diethylbutylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(9) 16β-(N-neopentylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(10) 16β-(N-2-adamantylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(11) 16β-N-1-adamantylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,,

(12) 16β-(N-2-norbomylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(13) 16β-(N-1-norbomylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(14) 16β-(N-phenylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(15) 16β-(N-benzylcarbamoyl)-4-oxa-5α-androst-1-en-3-one,

(16)16β-(2,3-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-oxa-5.alpha.-androst-1-en-3-one,

(17)16β-(2,4-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-oxa-5.alpha.-androst-1-en-3-one,

(18)16β-(2,6-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-oxa-5.alpha.-androst-1-en-3-one,

(19)16β-(2,3-bis(trifluoromethyl)phenylcarbamoyl)-4-oxa-androst-1-en-3-one,

(20)16β-(2,4-bis(trifluoromethyl)phenylcarbamoyl)-4-oxa-androst-1-en-3-one,

(21)16β-(2,6-bis(trifluoromethyl)phenylcarbamoyl)-4-oxa-androst-1-en-3-one,

(22) 16β-(N-n-octylcarbamoyl)-4-methyl-4-oxa-5α-androst-1-en-3-one,

(23) 16β-(1-methoxyethyl)-7β-methyl-4-oxa-5α-androst-1-en-3-one

(24) 16β-(isopropyl)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(25) 16β-(4-methyl-phenoxy)-7p-methyl-4-oxa-5α-androst-1-en-3-one,

(26)16β-(1-(3-chlorophenoxy)ethyl)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(27)16β-(4-methylsulfonyl-phenoxy)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(28) 16β-(4-chlorophenoxy)-7β-methyl-4-oxa-5α-androst-1-en-3-one,

(29) 16β-(2-pyrimidinyloxy)-7β-methyl-4-oxa-5α-androst-1-en-3-one, and

(30)16β-(2,5-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-oxa-5.alpha.-androst-1-en-3-one.

In one subclass of this class, R¹ is H or CH₃, R² is selected from H andCH₃, and A² is selected from: substituted and unsubstituted aryl orheteroaryl ether. Further illustrating this subclass are the compoundswherein substituted and unsubstituted aryl or heteroaryl ether isselected from thiophenoxy, biphenyloxy, acetamidophenoxy,(3-pyridyl)oxy, chlorophenyloxy, methylphenyloxy, phenoxy,hydroxyphenyloxy, methylsulfonylphenyloxy and pyrimidinyloxy. Stillfurther illustrating the compounds of the present invention arecompounds wherein substituted or unsubstituted aryl or heteroaryl etheris selected from 4-methyl-phenoxy, 4-chlorophenoxy, and2-pyrimidinyloxy.

In another embodiment of the compounds of structural formula (III) arecompounds of formula I wherein X is sulfur.

In one class of the compounds of this embodiment are compounds wherein Zis ##STR12##

Exemplifying compounds of this class are:

(1) N-t-Butyl-4-thia-5α-androst-1-en-3-one-17β-carboxamide,

(2) 7β-Methyl-4-thia-5α-cholest-1-en-3-one,

(3)17β-(2,5-bis(trifluoromethyl)phenylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(4)17β-(2,3-bis(trifluoromethyl)phenylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(5)17β-(2,4-bis(trifluoromethyl)phenylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(6)17β-(2,6-bis(trifluoromethyl)phenylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(7) 17β-(N-tert-amylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(8) 17β-(N-tert-hexylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(9) 17β-(N-isobutylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(10) 17β-(N-tert-octylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(11) 17β-(N-1,1-diethylbutylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(12) 17β-(N-neopentylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(13) 17β-(N-2-adamantylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(14) 17β-(N-1-adamantylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(15) 17β-(N-2-norbomylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(16) 17β-(N-1-norbornylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(17) 17β-(N-phenylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(18) 17β-(N-benzylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(19)17β-(2,3-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(20)17β-(2,4-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(21)17β-(2,6-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(22) 17β-(N-n-octylcarbamoyl)-4-methyl-4-thia-5α-androst-1-en-3-one,

(23) 17β-(1-methoxyethyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(24) 17β-(isopropyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(25) 17β-(4-methyl-phenoxy)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(26)17β-(1-(3-chlorophenoxy)ethyl)-7α-methyl-4-thia-5α-androst-1-en-3-one,

(27)17β-(4-methylsulfonyl-phenoxy)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(28) 17β-(4-chlorophenoxy)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(29) 17β-(2-pyrimidinyloxy)-7β-methyl-4-thia-5α-androst-1-en-3-one, and

(30)17β-(2,5-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-thia-5α-androst-1-en-3-one.

In one subclass of this class are compounds wherein R¹ is hydrogen, R²is selected from H and CH₃, and A¹ is selected from: carboxamide,including substituted and unsubstituted anilide derivatives.

Further illustrating this subclass are compounds wherein carboxamide isselected from: N,N-diisopropyl carboxamide, N-t-butyl carboxamide,N-t-octyl carboxamide, N-n-octyl carboxamide, N-(hydroxyphenyl)carboxamide, N-phenylcarboxamide, N-(aminophenyl) carboxamide,N-(carbomethoxy)phenyl carboxamide, N-(methoxycarboxy) phenylcarboxamide, N-acetamidophenyl-N-acetyl-carboxamide,N-acetamidophenyl-carboxamide, N-pivalamidophenyl carboxamide,N-isobutyramidophenyl carboxamide, N-(methyl),N-(diphenylmethyl)carboxamide, N-(diphenylmethyl)-carboxamide, N-t-butyl carboxamide,N-isopropyl carboxamide, 1-adamantyl carboxamide, 2-adamantylcarboxamide, and N-(substituted phenyl) carboxamides wherein the phenylmay be substituted with 1 to 2 substitutents selected from ethyl,methyl, trifluoromethyl or halo (F, Cl, Br, I).

In another subclass of this class are compounds wherein R¹ is CH₃, R² isselected from H and CH₃, and A¹ is a selected from: carboxamide,including substituted and unsubstituted anilide derivatives, and C₁₋₁₀alkyl. Further illustrating this subclass are compounds whereincarboxamide is --C(═O)NH--C₆ H₅ or --C(═O)NH-(2,5-trifluoromethylphenyl)and C₁₋₁₀ alkyl is selected from isopropyl, isobutyl, 1,5-dimethylhexyl,and 5-methylhexyl.

In another class of the compounds of this embodiment are compoundswherein Z is ##STR13##

Exemplifying compounds of this class are:

(1) N-t-Butyl-4-thia-5α-androst-1-en-3-one-16β-carboxamide,

(2) 16β-(1,5-dimethylhexyl)-7β-Methyl-4-thia-5α-androst-1-en-3-one,

(3)16β-(2,5-bis(trifluoromethyl)phenylcarbamoyl)-4-thia-androst-1-en-3-one,

(4) 16β-(N-tert-amylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(5) 16β-(N-tert-hexylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(6) 16β-(N-isobutylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(7) 16β-(N-tert-octylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(8) 16β-(N-1,1-diethylbutylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(9) 16β-(N-neopentylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(10) 16β-(N-2-adamantylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(11) 16β-(N-1-adamantylcarbamoyl)-4-thia-5α-androst-1-en-3-one,,

(12) 16β-(N-2-norbornylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(13) 16β-(N-1-norbornylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(14) 16β-(N-phenylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(15) 16β-(N-benzylcarbamoyl)-4-thia-5α-androst-1-en-3-one,

(16)16β-(2,3-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(17)16β-(2,4-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(18)16β-(2,6-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(19)16β-(2,3-bis(trifluoromethyl)phenylcarbamoyl)-4-thia-androst-1-en-3-one,

(20)16β-(2,4-bis(trifluoromethyl)phenylcarbamoyl)-4-thia-androst-1-en-3-one,

(21)16β-(2,6-bis(trifluoromethyl)phenylcarbamoyl)-4-thia-androst-1-en-3-one,

(22) 16β-(N-n-octylcarbamoyl)-4-methyl-4-thia-5α-androst-1-en-3-one,

(23) 16β-(1-methoxyethyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(24) 16β-(isopropyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(25) 16β-(4-methyl-phenoxy)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(26)16β-(1-(3-chlorophenoxy)ethyl)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(27)16β-(4-methylsulfonyl-phenoxy)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(28) 16β-(4-chlorophenoxy)-7β-methyl-4-thia-5α-androst-1-en-3-one,

(29) 16β-(2-pyrimidinyloxy)-7β-methyl-4-thia-5α-androst-1-en-3-one, and

(30)16β-(2,5-bis(trifluoromethyl)phenylcarbamoyl)-7β-methyl-4-thia-5α-androst-1-en-3-one.

In a subclass of this class, R¹ is H or CH₃, R² is selected from H andCH₃, and A² is selected from: substituted and unsubstituted aryl orheteroaryl ether. Further illustrating this subclass are the compoundswherein substituted and unsubstituted aryl or heteroaryl ether isselected from thiophenoxy, biphenyloxy, acetamidophenoxy,(3-pyridyl)oxy, chlorophenyloxy, methylphenyloxy, phenoxy,hydroxyphenyloxy, methylsulfonylphenyloxy and pyrimidinyloxy. Stillfurther illustrating the compounds of the present invention arecompounds wherein substituted or unsubstituted aryl or heteroaryl etheris selected from 4-methyl-phenoxy, 4-chlorophenoxy, and2-pyrimidinyloxy.

When any variable (e.g., aryl, heterocycle, R¹, etc.) occurs more thanone time in any constituent or in formula I, its definition on eachoccurrence is independent of its definition at every other occurrence.Also, combinations of substituents and/or variables are permissible onlyif such combinations result in stable compounds.

As used herein "alkyl" is intended to include both branched- andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, e.g., methyl (Me), ethyl (Et), propyl,butyl, pentyl, hexyl, heptyl, octyl, nonanyl, decyl, undecyl, dodecyl,and the isomers thereof such as isopropyl (i-Pr), isobutyl (i-Bu),secbutyl (s-Bu), tertbutyl (t-Bu), isopentane, isohexane, etc."Alkyloxy" (or "alkoxy") represents an alkyl group having the indicatednumber of carbon atoms attached through an oxygen bridge, e.g., methoxy,ethoxy, propyloxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy,t-butoxy and the like.

The term "aryl" includes phenyl and naphthyl. Preferably, aryl isphenyl.

Heteroaryl is selected from piperidinyl, piperizinyl, pyrrolidinyl,pyrrolyl, furanyl, thienyl, pyridyl, pyrimidinyl, indolyl andbenzofuranyl.

Heterocyclic rings may be attached to structural formula I at anyheteroatom (N, O or S) or carbon atom in the ring which results in thecreation of a stable, uncharged structure.

Hydroxy and amino protecting groups are known to those of ordinary skillin the art, and any such groups may be used. For example, acetate,benzoate, ether and silyl protecting groups are suitable hydroxyprotecting groups. Standard silyl protecting groups have the generalformula --Si(Xa)₃, wherein each Xa group is independently an alkyl oraryl group, and include, e.g. trimethylsilyl, tri-ethylsilyl,tri-i-propylsilyl, triphenylsilyl as well as t-butyl-di-(Xb)-silyl whereXb is methyl, ethyl, i-propyl or phenyl (Ph). Standard amino protectinggroups have the general formula --C(O)--Xc, wherein Xc is alkyl, aryl,O-alkyl or O-aryl, and include, e.g. N-t-butoxycarbonyl. See alsoProtective Groups in Organic Synthesis, T. W. Green et al. (John Wileyand Sons, 1991) for descriptions of protecting groups. The 4-oxacompounds of this invention can be prepared as shown in Scheme 2.##STR14##

Scheme 2 outlines the synthesis of the novel oxasteroids of the presentinvention. The appropriately substituted seco-acid may be prepared bymethods known in the art. PCT publication WO 95/11254 describesprocedures for the synthesis of compounds having various substituents atthe 16-position of the azasteroid. Starting with a 3-keto-delta4-17-oneprecursor and following the procedures of WO 95/11254, the appropriateA² substitution may be obtained. To obtain the appropriate A¹substitution, the procedures of the following publications are followedstarting with a 3-keto-delta4-17-one precursor: for ether or thioetherWO 93/23040; for anilide WO 94/07861, EP 0 663 924; for unsubstituted,monosubstituted or disubstituted amides WO 93/23038, WO 93/23051, WO93/23420; and U.S. Pat. Nos. 4,220,775, 4,760,071, 4,845,104, 5,237,067,5,091,380, 5,061,801, 5,215,894, for oxo U.S. Pat. Nos. 4,220,775,4,377,584; for cyano U.S. Pat. No. 4,220,775; for tetrazoyl U.S. Pat.No. 4,220,775; for arylalkylcarbonlyoxy alkyl U.S. Pat. No. 4,377,584;for cycloalkylarylcarbonlyoxy alkyl U.S. Pat. No. 4,377,584; forbenzoyloxyalkyl U.S. Pat. No. 4,377,584; for acyl, both substituted andunsubstituted, U.S. Pat. No. 5,049,562, U.S. Pat. No. 5,138,063, U.S.Pat. No. 5,151,429, U.S. Pat. No. 5,237,061, U.S. Pat. No. 5,120,742,U.S. 5,162,332, U.S. Pat. No. 5,061,802, U.S. Pat. No. 5,098,908, U.S.Pat. No. 5,196,411, U.S. 5,075,450, U.S. Pat. No. 5,061,803, U.S. Pat.No. 5,324,734; for thiobenzyl U.S. Pat. No. 5,151,430; for polyaroylU.S. Pat. No. 5,162,322; for ester U.S. Pat. No. 5,091,534, WO 93/23041,WO 93/23040, for alkyl, either substituted or unsubstituted WO 93/23050,WO 93/23419, WO 93/23051; for urea, thiourea, carbamate or thiocarbamateW093/23048; for thioester WO 93/23041, WO 93/23040.

The appropriately 7,10,16, and 17-substituted 3-keto steroid isconverted to the appropriately substituted seco-acid by methods known inthe art, for example, the procedures described in Rasmusson, et al., J.Med. Chem. 1986, 29(11): 2298-2315.

The appropriate 7-βsubstitution may be obtained following the proceduresfor formation of a 7-βbond as described in U.S. Pat. Nos. 4,220,775, and5,237,064.

Compounds wherein R² is H or CH₂ OR³ may be prepared starting with theappropriately C10 substituted seco-acid. These compounds may be made byprocedures known in the art.

As shown in Scheme 2, the seco-acid (1) is treated with a dehydratingagent such as acetic anhydride, methyl ortho-formate, ethylortho-formate, in a nonpolar aprotic solvent such as toluene, xylene,dichloroethane, chlorobenzene and the like optionally in the presence ofan acidic catalyst, such as PTSA (paratoluenesulfonic acid), or sodiumacetate to form the Δ⁵ -oxasteroid (2). Preferably, the seco-acid (1) istreated with acetic anhydride in acetic anhydride in the presence ofsodium acetate at an elevated temperature, preferably at about 140° C.Hydrogenation of the double bond to form the oxasteroid (3) may becarried out in the presence of an appropriate catalyst such as Rh/C,Pd/C, etc., preferably Rh/C in a solvent such as tetrahydrofuran (THF)or ethyl acetate. This is followed by formation of the Δ¹ double bond bytreatment with, for example, dichloro-dicyanoquinone (DDQ),benzeneselenic anhydride in chlorobenzene, or other known methods, forexample as described in U.S. Pat. Nos. 5,084,574 and 5,021,571, to formthe Δ¹ -oxasteroid (4).

The 4-thia compounds of this invention can be prepared as shown inScheme 3. ##STR15##

Starting with the 4-oxa-androstan-3-one appropriately substituted in theB and D-ring, obtained by following the procedures of Scheme 2 to obtaincompound (3), the lactone is opened to form the hydroxamide (5). Thelactone may be opened by various means such as treatment withdimethylalumino-3-aminopyridine (which may be prepared in situ bytreating trimethyl aluminum with 3-amino pyridine), chlorobenzene ordichloroethane in a nonpolar, aprotic solvent such as toluene.

The hydroxamide (5) is treated with alkyl- or aryl sulfonyl chloride ina solvent such as methylene chloride, toluene or dichloroethane in thepresence of a base such as pyridine, dimethylaminopyridine (DMAP), orN-methylpyrolidine (NMP), to give the corresponding alkyl- or arylsulfonate (6). The reaction of the alkyl- or aryl sulfonate withtetrabutylammonium iodide in a polar aprotic solvent such as tolueneproduces the corresponding iodide (7). The iodide is treated withthioacetic acid in a nonpolar solvent such as toluene or dichloroethanein the presence of cesium carbonate or other base such as potassiumcarbonate or sodium carbonate to give the thioacetate (8). Thethioacetate (8) is hydrolyzed to form the thialactone (9), preferably bytreatment with acid in a polar solvent such as methanol or ethanol,preferably by treatment with hydrochloric acid in methanol. Thethialactone (9) may be dehydrogenated to form the Δ¹ -thiasteroid (10)as described above, preferably by treatment with benzeneselenicanhydride in chlorobenzene at reflux.

The 4-oxa and 4-thia steroids of the structural formula (III) includethe 1,2-5,6 diene which may be prepared by treating the compound ofstructural formula (2) with benzeneselenic anhydride in chlorobenzenewith refluxing to obtain the Δ¹,Δ⁵ -oxasteroid derivative. Thecorresponding thiasteroid derivative may be obtained by following theprocedures of Scheme 2, starting with (2), the Δ⁵ -oxasteroid.

More particularly, the present invention relates to a method fortreating hyperandrogenic conditions in a human being in need of suchtreatment by irreversibly inhibiting the human 5α-reductase enzymewithout covalently modifying the 5α-reductase enzyme. This methodcomprises the administration to the human in need of such treatment of a3-oxo-4-oxa and 4-thiasteroid having a 1,2-double bond. The 3-oxo-4-oxaand 4-thiasteroid having a 1,2-double bond is selectively activated by5α-reductase to produce the covalent adduct with the cofactor NADPH ofstructural formula (I). ##STR16## wherein X is selected from oxygen andsulfur. This covalent adduct is released so slowly from the 5α-reductaseenzyme that the inhibition of the enzyme is effectively irreversible.

The method of the present invention provides for an inhibitor whichdisplays the characteristics of a suicide inhibitor without covalentlymodifying the enzyme. Generally, "suicide inhibitors" are inhibitorsthat deactivate the enzyme by covalent modification of the enzymeprotein. Such "suicide inhibitors" are not favored as pharmaceuticalagents because the covalently-modified enzyme may be recognized by theimmune system of the treated organism as foreign matter and trigger anundesirable immunological response. The non-protein bound adduct of thepresent invention eliminates the possibility of such an undesirableimmunological response.

The long-lived enzyme-bound 3-oxo-4-oxasteroid-NADP adduct and3-oxo-4-thiasteroid NADP adduct of the present invention providesfurther advantages in clinical settings. The method of the presentinvention provides for sustained inhibition of 5α-reductase even whenthe dose regimen is interrupted and the levels of the 3-oxo-4-oxasteroidor 3-oxo-4-thiasteroid drug having a 1,2-double bond drop, as when thepatient misses a dose. In this situation of interrupted dosing,5α-reductase that had been inhibited cannot recover from the inhibitionand additional drug is required to inhibit only the 5α-reductase thathas been newly synthesized by the patient.

Hyperandrogenic conditions treatable by the method of the presentinvention include benign prostatic hyperplasia, androgenic alopecia,acne vulgaris, seborrhea, female hirsutism, prostatitis and prostaticcarcinoma.

The 3-oxo-4-oxa and 4-thiasteroids having a 1,2-double bond useful inthe present invention are typically administered in admixture withsuitable pharmaceutical diluents, excipients or carriers (collectivelyreferred to herein as "carrier" materials) suitably selected withrespect to the intended form of administration, that is, oral tablets,capsules, elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices may be administered systemically, by oraladministration or by intravenous or intramuscular injection ortopically.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Capsules containing the product of thisinvention can be prepared by mixing an active compound of the presentinvention with lactose and magnesium stearate, calcium stearate, starch,talc, or other carriers, and placing the mixture in gelatin capsules.

Tablets may be prepared by mixing the active ingredient withconventional tableting ingredients such as calcium phosphate, lactose,corn starch or magnesium stearate. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders includestarch, gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes andthe like. Lubricants used in these dosage forms include sodium oleate,sodium stearate, magnesium stearate, sodium benzoate, sodium acetate,sodium chloride and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gum andthe like.

The liquid forms in suitably flavored suspending or dispersing agentssuch as the synthetic and natural gums, for example, tragacanth, acacia,methyl-cellulose and the like. Other dispersing agents which may beemployed include glycerin and the like. For parenteral administration,sterile suspensions and solutions are desired. Isotonic preparationswhich generally contain suitable preservatives are employed whenintravenous administration is desired.

Topical pharmaceutical compositions may be, e.g., in the form of asolution, cream, ointment, gel, lotion, shampoo or aerosol formulationadapted for application to the skin. Topical pharmaceutical compositionsuseful in the method of treatment of the present invention may includeabout 0.001% to 0.1% of the active compound in admixture with apharmaceutically acceptable carrier.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, propylene glycol, PPG2 myristyl propionate, and thelike, to form, e.g., alcoholic solutions, topical cleansers, cleansingcreams, skin gels, skin lotions, and shampoos in cream or gelformulations. See, e.g., EP 0 285 382.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds of the present invention may alsobe coupled with soluble polymers as targetable drug carriers. Suchpolymers can include polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamide-phenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

Advantageously, the active agent of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in dividend doses of two, three or four times daily. Thecompounds for the present invention can be administered in intranasalform via topical use of suitable intranasal vehicles, or via transdermalroutes, using those forms of transdermal skin patches well known tothose of ordinary skill in the art. To be administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.Compounds of the present invention may also be delivered as asuppository employing bases such as cocoa butter, glycerinated gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights and fatty acid esters of polyethylene glycol.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularcompound thereof employed. A physician or veterinarian of ordinary skillcan readily determine and prescribe the effective amount of the drugrequired to prevent, counter, arrest or reverse the progress of thecondition. Optimal precision in achieving concentration of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug. Preferably, doses of the 3-oxo-4-oxa and4-thiasteroid having a 1,2-double bond useful in the method of thepresent invention range from 0.001 to 100 mg per day, preferably 0.05 to50 mg per day and as provided by the advantage of the present invention,doses inadvertently missed will not compromise the therapeutic efficacy.Most preferably, dosages range from 0.01 to 10 mg/day.

The Δ¹ -3-oxo-4-oxa and 4-thiasteroids of the present invention may beadministered on a cyclical regimen. The details of the effective regimendepend on the particular Δ¹ -3-oxo-4-oxa and 4-thiasteroid administered.These cyclical regimens provide an advantage over classical drugs, i.e.,pharmaceutically active agents that do not function as an irreversibleinhibitor.

The Δ¹ -3-oxo-4-oxa and 4-thiasteroids of the present invention may beadministered in the form of pharmaceutically acceptable salts. The term"pharmaceutically acceptable salt" is intended to include all acceptablesalts such as hydrochloride, hydrobromide, acetate, panoate and the likewhich can be used as a dosage form for modifying the solubility orhydrolysis characteristics or can be used in sustained release orpro-drug formulations. The term "pharmaceutically acceptable salt" isintended to include all acceptable salts such as acetate, lactobionate,benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate,bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide,bromide, methylnitrate, calcium edetate, methylsulfate, camsylate,mucate, carbonate, napsylate, chloride, nitrate, clavulanate,N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate,edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate,esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate,polygalacturonate, gluconate, salicylate, glutamate, stearate,glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine,succinate, hydrobromide, tanmate, hydrochloride, tartrate,hydroxynaphthoate, teoclate, iodide, tosylate, isothionate,triethiodide, lactate, panoate, valerate, and the like which can be usedas a dosage form for modifying the solubility or hydrolysischaracteristics or can be used in sustained release or pro-drugformulations. Depending on the particular functionality of the compoundof the present invention, pharmaceutically acceptable salts of thecompounds of this invention include those formed from cations such assodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and frombases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,arginine, ornithine, choline, N,N'-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)aminomethane, andtetramethylammonium hydroxide. These salts may be prepared by standardprocedures, e.g. by reacting a free acid with a suitable organic orinorganic base.

Also, in the case of an acid (--COOH) or alcohol group being present,pharmaceutically acceptable esters can be employed, e.g. acetate,maleate, pivaloyloxymethyl, and the like, and those esters known in theart for modifying solubility or hydrolysis characteristics for use assustained release or prodrug formulations.

The compounds of the present invention may have chiral centers otherthan those centers whose stereochemistry is depicted in formulae I, IIand III, and therefore may occur as racemates, racemic mixtures and asindividual enantiomers or diastereomers, with all such isomeric formsbeing included in the present invention as well as mixtures thereof.Furthermore, some of the crystalline forms for compounds of the presentinvention may exist as polymorphs and as such are intended to beincluded in the present invention. In addition, some of the compounds ofthe instant invention may form solvates with water or common organicsolvents. Such solvates are encompassed within the scope of thisinvention.

For the treatment of acne vulgaris, androgenic alopecia, male patternbaldness, seborrhea, female hirsutism, benign prostatic hyperplasia,prostatitis and the prevention and/or treatment of prostatic cancer, thecompounds of the instant invention can be combined with atherapeutically effective amount of another 5α-reductase inhibitor, suchas finasteride or epristeride, or other 5α-reductase inhibitor compoundshaving type 2 activity, type 1 activity or dual activity for bothisozymes, in a single oral, systemic, or parenteral pharmaceuticaldosage formulation. Alternatively, a combined therapy can be employedwherein the compound of formula I and the other 5α-reductase inhibitorare administered in separate oral, systemic, or parenteral dosageformulations. Also, for the skin and scalp related disorders of acnevulgaris, androgenic alopecia, male pattern baldness, seborrhea, andfemale hirsutism, the compounds of the instant invention and another5α-reductase inhibitor such as finasteride or epristeride can beformulated for topical administration. For example, a compound offormula I and finasteride can be administered in a single oral ortopical dosage formulation, or each active agent can be administered ina separate dosage formulation, e.g., in separate oral dosageformulations, or an oral dosage formulation of finasteride incombination with a topical dosage formulation of a compound of formulaI. See, e.g., U.S. Pat. Nos. 4,377,584 and 4,760,071 which describedosages and formulations for 5α-reductase inhibitors.

Furthermore, administration of a compound of the present invention incombination with a therapeutically effective amount of a potassiumchannel opener, such as minoxidil, cromakalin, pinacidil, a compoundselected from the classes of S-triazine, thiane-1-oxide, benzopyran, andpyridinopyran derivatives or a pharmaceutically acceptable salt thereof,may be used for the treatment of androgenic alopecia including malepattern baldness. Therapy may further comprise the administration of a5α-reductase type 2 inhibitor such as finasteride or epristeride, or a5α-reductase type 1 inhibitor, or a type 1 and type 2 dual inhibitor, incombination with a compound of the present invention and a potassiumchannel opener such as minoxidil. The active agents can be administeredin a single topical dosage formulation, or each active agent can beadministered in a separate dosage formulation, e.g., in separate topicaldosage formulations, or an oral dosage formulation of a compound offormula I in combination with a topical dosage formulation of, e.g.,minoxidil, or a single oral dosage formulation of a compound of formulaI and another 5α-reductase inhibitor, in combination with a topicaldosage formulation of, e.g., minoxidil. See, e.g., U.S. Pat. Nos.4,596,812, 4,139,619 and WO 92/02225, published 20 Feb. 1992, fordosages and formulations of calcium channel openers.

Furthermore, for the treatment of acne vulgaris, a combined therapy canbe used by administering a therapeutically effective amount of acompound of formula I in combination with a therapeutically effectiveamount of retinoic acid or a derivative thereof, e.g. an ester or amidederivative thereof, such as e.g., tretinoin or isotretinoin. Optionally,this combined therapy for acne vulgaris may further include a5α-reductase type 2 inhibitor such as finasteride or epristeride, or a5α-reductase type 1 inhibitor, or a dual type 1 and type 2 inhibitorycompound.

Also, for the treatment of benign prostatic hyperplasia, a combinedtherapy comprising a administration of a compound of formula I with a5α-reductase type 2 inhibitor, such as e.g., finasteride, and an alpha-1adrenergic receptor antagonist, such as e.g., terazosin, doxazosin,prazosin, bunazosin, indoramin or alfuzosin, may be employed. Moreparticularly, the combined therapy can comprise administering a compoundof formula I with a 5α-reductase type 2 inhibitor, such as e.g.,finasteride, and an alpha-1_(a) adrenergic receptor antagonist (formerlycalled an alpha-1_(c) adrenergic receptor antagonist). Compounds whichare useful as alpha-1_(a) adrenergic receptor antagonists can beidentified according to procedures known to those of ordinary skill inthe art, for example, as described in PCT/US93/09187 (W094/08040,published Apr. 14, 1994); PCT/US94/03852 (WO 94/22829, published Oct.13, 1994); PCT/US94/10162 (WO 95/07075, published Mar. 16, 1995), andU.S. Pat. No. 5,403,847.

Also, for the treatment of acne vulgaris, androgenic alopecia,seborrhea, female hirsutism, benign prostatic hyperplasia, prostatitisand the prevention and/or treatment of prostatic cancer, a combinedtherapy can be used by administering a therapeutically effective amountof a compound of formula I with a therapeutically effective amount of ananti-androgen, such as, e.g., flutamide, spironolactone or casodex.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concomitantly, or they each can be administered atseparately staggered times.

Biological Assays

The standard assay contains 100 pM membrane-bound enzyme suspended in asolution of 25 nM ³ H!testosterone (139,000 dpm, carrier free) and 500μM NADPH in a buffer consisting of 0.1M MOPS, 1 mM EDTA, and 0.1% BSA,at pH 7.20 and 37° C., in a total volume of 100 μL. Ethanol is includedat 1% final concentration as the vehicle for introduction of inhibitors.The rate of production of ³ H!dihydrotestosterone is approximatelyconstant for up to 2 hours or 10% consumption of substrate. A unit ofenzyme activity was defined as 1 pmol product/min, and is equal to 444fmol enzyme under these conditions.

Alternatively, measurements may be conducted under conditions that havemore traditionally been employed for this enzyme at pH 5.50 and 37° C.

After quenching the reactions with an equal volume, or more, of watercontaining 1% trifluoroacetic acid, the ³ H!dihydrotestosterone isisolated by direct injection onto a reverse phase C-18 column (Vydac,4.6×250 mm, 300 Å, 5 micron), which is run in an isocratic system of 40%water (containing 0.1% trifluoroacetic acid) and 60% methanol at 1mL/min. When tissue extracts are the source of enzyme, the quenchedreactions are clarified by centrifugation at 10,000×g before analysis.Retention times are ˜13 min for testosterone and ˜20 min fordihydrotestosterone. The effluent containing the ³ H!dihydrotestosteronepeak (6 mL) is collected in a liquid scintillation vial and counted withAQUASOL 2 (14 mL, New England Nuclear) with an efficiency of 0.309. Theassay is completely automated using a SUN SPARKSTATION 2 computerinterfaced to a ZYMARK robot and ancillary equipment.

Enzyme Sources

The native human enzyme was from prostate (type 2) or scalp (type 1)tissue, and the recombinant human enzymes were produced by thebaculovirus expression system of Andersson, et al. (Chan, H. K.,Geissler, W. M., Andersson, S., Sex Hormones and Antihormones inEndocrine Dependent Pathology: Basic and Clinical Aspects, M. Motta andM. Serio, eds., Elsevier Science 1994). A comparable baculovirusexpression system also has been described recently by Tian et al.,Biochemistry, 33: 2291-2296 (1994). Enzyme concentrations weredetermined by titration with finasteride, or ³ H!finasteride, or fromcatalytic activity using kcat and Km values.

Under the standard assay conditions, the K_(m) of thebaculovirus-expressed type 2 enzyme for testosterone was found to be24.6±0.7 nM at pH 7.2 and 37° C. This K_(m) value agrees with thatdetermined by Faller, et al. Biochemistry 32: 5705-5710 (1993) for thenative human prostate enzyme at neutral pH (K_(m) =20±3 nM). The K_(m)for NADPH was found to be ˜1 μM under the assay conditions and 10×K_(m)testosterone. The turnover number was k_(cat) =0.075±0.006 s⁻¹ at pH 7.2and 37° C., as determined by titration with finasteride. From thesekinetic constants, k_(cat) /K_(m) =2.99±0.32×10⁶ M⁻¹ s⁻¹. The K_(m) forthe type 1 5α-reductase was 7 μM, and the kcat for the type 1 enzyme was1.4 sec⁻¹. The molecular weight was taken to be about 30,000 inestimating enzyme purity.

Inhibition studies

Compounds were dissolved in 100% ethanol. The compound to be tested waspre-incubated with the enzyme (either 5α-reductase type 1 or 2) prior toinitiation by addition of substrate testosterone. IC₅₀ values representthe concentration of inhibitor required to decrease enzyme conversion oftestosterone to dihydrotestosterone by 50% of the control. IC₅₀ valueswere determined using a 6 point titration where the concentration of theinhibitor was varied from 0.1 to 1000 nM. Representative compounds ofthis invention were tested in the above described assay for 5α-reductasetype 1 and type 2 inhibition.

A compound referred to herein as a 5α-reductase 1 inhibitor is acompound that shows inhibition of the 5α-reductase 1 isozyme in theabove-described assay, having an IC₅₀ value of about or under 100 nM.

A compound referred to herein as a 5α-reductase 2 inhibitor is acompound that shows inhibition of the 5α-reductase 2 isozyme in theabove-described assay, having an IC₅₀ value of about or under 100 nM.

EXAMPLE 1 Determination of Rate Constant for Formation of The Enzyme:17β-(N-diethyl)carbamoyl-4-oxa-5α-androst-1-en-3-one Complex

Progress curves of DHT formation were used to follow the time course forinhibition by (17β-(N-diethyl)carbamoyl-4-oxa-5α-androst-1-en-3-one(L-682,299). For this example, baculovirus expressed type 2 5α-reductasewas incubated with 25 nM ³ H!testosterone and 100 μM NADPH with orwithout 20 nM inhibitor in a buffer composed of 0.1M MOPS, 0.1% BSA, and1 mM EDTA at pH 7.2 and 37° C. Aliquots were withdrawn and testosteroneseparated from DHT by reverse phase HPLC (C18 column 1 mL/min, 60%methanol, 0.1% TFA, T elutes at about 13 minutes, DHT at about 20minutes. The ³ H in the T and DHT fractions was determined byscintillation counting using an in line radioflow detector.Alternatively, fractions may be collected and the samples counted on ascintillation counter. With this 4-oxasteroid, the inhibition developedslowly with time and resulted in total inhibition of the enzyme atinfite time. The time courses were fit to an integrated first-order rateequation (Equation 1), where A is product concentration, v_(o) is theinitial velocity, v_(s) is the infinite-time velocity, and k_(obs) isthe first-order rate constant for progression of the enzyme between thetwo equilibrium states. This 4-oxasteroid was found to bind to the type2 isozyme with a second-order rate constant of 5.60×10⁴ M⁻¹ s⁻¹. Thevelocity at infinite-time was essentially zero. Consequently, theinhibitor was determined to be extremely potent, having a Ki<<100 pM.(Ki=k_(off) /k_(on)).

    A=v.sub.s t+(v.sub.o -v.sub.s)(1-e-.sup.k obs.sup.t)/k.sub.obs +Ao Equation 1

EXAMPLE 2 Determination of Rate Constant for Formation of The Enzyme:Inhibitor Complex

Similar studies may be conducted as described in Example 1 with other4-oxa or 4-thia steroids having a 1,2 double bond using either the type1 or type 2 5α-reductase. The enzyme used in these experiments may beeither from native sources (human scalp or prostate) or recombinantlyproduced (baculovirus-expressed, COS cell expressed).

EXAMPLE 3 Determination of The Rate of Release of The Inhibitor From TheEnzyme

Starting with radioactively-labeled inhibitor such as 1,2-³H!-inhibitor, which may be prepared according to procedures well knownin the art, such as tritium reduction of the Δ¹ precursor with tritiumgas and a Pd/C catalyst as described by Liang et al. Endocrinology(1983) 112: 1430, the loss of the ³ H from the enzyme could be used todetermine the rate of release of the inhibitor from the type 1 or 25α-reductase. Either native or recombinantly-expressed 5α-reductase maybe incubated with the 1,2-³ H!-inihibitor and NADPH. If necessary,excess ³ H-inhibitor not bound to the enzyme may be removed by dialysisprior to determination of the radioactivity bound to the enzyme. Theconcentrations of bound and free radioactivity could be determined byultrafiltration on 10,000 Da cutoff membranes (AMICON, Centricon-10),which would be centrifuged for several hours at 4° C. Typically, 100 μLsamples would be diluted to 1 mL with water before ultrafiltration, andthe 3H in the total solution and the filtrate determined byscintillation counting.

EXAMPLE 4 Evidence For Reduction of The Inhibitor

Type 1 or 2 5α-reductase (native or recombinantly produced) could beincubated with radiolabeled inhibitor and NADPH to produce theenzyme:inhibitor complex. Excess inhibitor could be removed by dialysisof the enzyme solution. Release of the reduced inhibitor from theenzyme:inhibitor complex can be accomplished by heat denaturation in aboiling water bath for 90 min. The denatured protein can be removed bycentrifugation or ultrafiltration through a 10,000 Da cutoff membrane(AMICON, YM-10). The identity of the radioactive species could bedetermine d by reverse phase high pressure liquid chromatography, forexample a C₈ column with a linear gradient of 1 mL/minute of water to100% methanol over 30 minutes.

The material released from the enzyme will no longer comigrate with theparent Δ¹ -4-oxa or 4-thia inhibitor. This process could be scaled up toproduce sufficient material for structure identification by massspectrometry.

EXAMPLE 5 Evidence For Noncovalent Binding to The Enzyme

Starting with radiolabeled inhibitor such as 1,2-³ H! inhibitor,enzyme:inhibitor complex could be prepared by incubation of the labeledinhibitor, NADPH and recombinantly produced or native type 1 or 25α-reductase. Denaturation of the enzyme-inhibitor complex with an equalvolume of 95% ethanol containing 20 mM ammonium bicarbonate, pH 9.0would liberate the NADP-inhibitor covalent adduct (structure 1). Thesuspension should be stirred for 30 min at room temperature, thencentrifuged at 10,000×g for 45 min to remove the insoluble matter.Alternatively, 6M guanidine:HCl or other organic solvents could be usedto liberate the adduct. After release of the adduct from the enzyme, allsolutions should contain ammonium bicarbonate adjusted to pH 9 withammonium hydroxide to buffer the free adduct. The radiolabeled adductcould be purified by using anion exchange chromatography such as aPharmacia Mono Q anion exchange column HCO-₃ form equilibrated to 0.01Mammonium bicarbonate, pH 9, and 50% methanol.

EXAMPLE 6 Preparation ofN-t-Butyl-4-oxa-5α-androst-1-en-3-one-17β-carboxamide

Step 1: Benzotriazol-1'-yl-3-oxo-androst-4-ene-17β-carboxamide.##STR17##

To a solution of steroid acid (4g, 12.65 mmol) in methylene chloride wasadded DCC (2.75 g, 13.3 mmol) and 1-hydroxybenzotriazole (HOBT, 2.75 g,19.1 mmol). After stirring the reaction mixture for overnight, the solidwas filtered, dried and used as such for further reactions.

Step 2: N-t-Butyl-3-oxo-androst-4-ene-17β-carboxamide. ##STR18##

To a solution of benzotriazol-1'-yl-3-oxo-androst-4-ene-17β-carboxamide(1.5 g, 3.46 mmol) in methylene chloride was added t-butyl amine (454μl, 4.33 mmol). After stirring the reaction mixture for overnight, thereaction mixture was concentrated, purified by chromatography oversilica gel (2.5% acetone/methylene chloride). Mass spec. M⁺ 372(m+1,observed).

Step 3: N-t-Butyl-5-oxo-3,5-secoandrostan-3-oic-17β-carboxamide.##STR19##

To a solution of N-t-butyl-3-oxo-androst-4-ene-17β-carboxamide (550 mg,1.48 mmol) in t-butanol (10 mL) was added sodium carbonate (172 mg, 1.63mmol, in 1 mL of H₂ O). The reaction mixture was heated to 80° and asolution of NaIO₄ (1.58 g, 7.4 mmol) and KMnO₄ (11.7 mg, 0.074 mmol) inH₂ O (10 mL) was added dropwise in ˜10 minutes. After stirring thereaction mixture for 2 hrs, the mixture was cooled to room temperatureand acidified to pH 2. The reaction mixture was concentrated, extractedwith ethyl acetate, organic layer was dried and concentrated to givepure product. Mass spec. M⁺ 392(m+1, observed).

Step 4: N-t-Butyl-4-oxa-androst-5-en-3-one-17β-carboxamide. ##STR20##

To a solution of N-t-butyl-5-oxo-3,5-secoandrostan-3-oic-17β-carboxamide(550 mg, 1.4 mmol) in acetic anhydride (25 mL) was added sodium acetate(1.91 g, 14 mmol). After stirring the reaction mixture at refluxtemperature for 4 hrs, the acetic anhydride was removed under vacuum andresidue partitioned between ethyl acetate and water. The organic layerwas washed with brine, dried, concentrated and purified by prep. tlc (5%acetone/methylene chloride). Mass spec. M⁺ 374(m+1, observed).

Step 5: N-t-Butyl-4-oxa-5α-androstan-3-one-17β-carboxamide. ##STR21##

To a solution of N-t-butyl-4-oxa-androst-5-en-3-one-17β-carboxamide (410mg, 1.099 mmol) in THF (20 mL) was added 5% Rh/C(410 mg). After stirringthe reaction mixture under H₂ atmosphere for overnight, the mixture wasflushed with N₂, filtered and concentrated. The residue was purified byprep. tlc (5% acetone/CH₂ Cl2). Mass spec. M⁺ 376(m+1, observed).

Step 6: N-t-Butyl-4-oxa-5α-androst-1-en-3-one-17β-carboxamide. ##STR22##

To a solution of N-t-butyl-4-oxa-5α-androstan-3-one-17β-carboxamide (100mg, 0.27 mmol) in chlorobenzene was added benzeneseleninic anhydride(177 mg, 0.49 mmol). After stirring the reaction mixture at refluxtemperature for overnight, the reaction mixture was diluted with ethylacetate, washed with brine, dried and concentrated. The residue waspurified by prep. tlc (5% acetone/CH₂ Cl₂). Mass spec. M⁺ 374(m+1,observed).

EXAMPLE 7 Preparation of 7β-Methyl-4-oxa-5α-Cholest-1-en-3-one

Step 1: 7β-Methyl-4-oxa-Cholest-5-en-3-one ##STR23##

To a solution of 7β-methyl-5-oxo-3,5-sec-cholestan-3-oic (5 g, 12.3mmol) in acetic anhydride (250 mL) was added sodium acetate (16.9 g, 124mmol). After stirring the reaction mixture at reflux temperature for 4hrs, the acetic anhydride was removed under vacuum and residuepartitioned between ethyl acetate and water. The organic layer waswashed with brine, dried, concentrated and purified by chromatographyover silica gel. Mass spec. M⁺ 401(m+1, observed).

Step 2: 7β-Methyl-4-oxa-5α-Cholestan-3-one ##STR24##

To a solution of 7β-methyl-4-oxa-Cholest-5-en-3-one (6.0 g) in THF (50mL) was added 5% Rh/C(6.5 g). After stirring the reaction mixture underH₂ atmosphere for overnight, the mixture was flushed with N₂, filteredand concentrated. The residue was purified by chromatography over silicagel (20% ethyl acetate/hexane). Mass spec. M⁺ 403(m+1, observed).

Step 3: 7β-Methyl-4-oxa-5α-Cholest-1-en-3-one ##STR25##

To a solution of 7β-methyl-4-oxa-5α-Cholestan-3-one (1 g, 2.49 mmol) inchlorobenzene (50 mL) was added benzeneseleninic anhydride (1.165 g,3.24 mmol). After sting the reaction mixture at reflux temperature forovernight, the reaction mixture was diluted with ethyl acetate, washedwith brine, dried and concentrated. The residue was purified bychromatography over silica gel (10% ethyl acetate/hexane). Mass spec. M⁺401 (m+1, observed).

EXAMPLE 8 Preparation ofN-(2',5'-Bistrifluoromethylphenyl)-4-oxa-androst-1-en-3-one-17β-carboxamide

Step 1 S-2'-Pyridyl-3-oxo-androst-4-ene-17β-thiocarboxylate ##STR26##

To a solution of steroid acid (10.26 g, 30 mmol) in toluene (50 mL) wasadded Aldrithiol (15.97 g, 72.49 mmol) and triphenylphosphine (16.88 g,72.49 mmol). After stirring the reaction mixture for overnight at 23°,the reaction mixture was concentrated and purified by chromatographyover silica gel using methylene chloride as solvent.

Step 2:N-(2',5'-Bistrifluoromethylphenyl)-3-oxo-androst-4-ene-17β-carboxamide##STR27##

To a solution of S-2'-pyridyl-3-oxo-androst-4-ene-17β-thiocarboxylate(2.179 g, 5.19 mmol) in methylene chloride was added2,5-bistrifluoromethylaniline (3 g, 13 mmol) and silver triflate (1.336g, 5.2 mmol). After stirring the reaction mixture for overnight at 23°,he mixture was filtered, concentrated and purified by chromatographyover silica gel using methylene chloride as solvent to give pureproduct.

Step 3:N-(2',5'-Bistrifluoromethylphenyl)-5-oxo-3,5-secoandrostan-3-oic-17β-carboxamide##STR28##

To a solution ofN-(2',5'-Bistrifluoromethylphenyl)-3-oxo-androst-4-ene-17β-carboxamide(600mg, 1.27 mmol) in t-butanol (10 mL) was added sodium carbonate (200 mg,1.88 mmol, in 1 mL of H₂ O). The reaction mixture was heated to 80° anda solution of NaIO₄ (1.81 g, 8.5 mmol) and KMnO₄ (13.2 mg, 0.08 mmol) inH₂ O (10 mL) was added dropwise in ˜10 minutes. After stirring thereaction mixture for 2 hrs, the mixture was cooled to room temperatureand acidified to pH 2. The reaction mixture was concentrated, extractedwith ethyl acetate, organic layer was dried and concentrated to givepure product

Step 4:N-(2',5'-Bistrifluoromethylphenyl)-4-oxa-androst-5-en-3-one-17β-carboxamide##STR29##

To a solution ofN-(2',5'-bistrifluoromethylphenyl)-5-oxo-3,5-secoandrostan-3-oic-17β-carboxamide(600 mg, 1.19 mmol) in acetic anhydride (10 mL) was added sodium acetate(1.63 g, 11.9 mmol). After stirring the reaction mixture at refluxtemperature for 4 hrs, the acetic anhydride was removed under vacuum andresidue partitioned between ethyl acetate and water. The organic layerwas washed with brine, dried, concentrated and purified by prep. tlc (5%acetone/methylene chloride). Mass spec. M⁺ 530 (m+1, observed).

Step 5:N-(2',5'-Bistrifluoromethylphenyl)-4-oxa-androstan-3-one-17β-carboxamide##STR30##

To a solution ofN-(2',5'-bistrifluoromethylphenyl)-4-oxa-androst-5-en-3-one-17β-carboxamide(230 mg, 0.433 mmol) in THF (5 mL) added 5% Rh/C (200 mg). Afterstirring the reaction mixture for overnight under hydrogen atmosphere,the reaction mixture was flushed with nitrogen, filtered, concentratedand purified by preparative tlc (50% EtOAc/hexane) to give pare product.Mass spec. M⁺ 532 (m+1, observed).

Step 6:N-(2',5'-Bistrifluoromethylphenyl)-4-oxa-androst-1-en-3-one-17β-carboxamide##STR31##

To a solution ofN-(2',5'-bistrifluoromethylphenyl)-4-oxa-androstan-3-one-17β-carboxamide.(35 mg, 0.066 mmol) in chlorobenzene (5 mL) was added benzeneseleninicanhydride (30 mg, 0.085 mmol). After stirring the reaction mixture atreflux temperature for overnight, the reaction mixture was diluted withethyl acetate, washed with brine, dried and concentrated. The residuewas purified by prep. tlc (50% EtOAc/hexane). Mass spec. M⁺ 530(m+1,observed).

EXAMPLE 9 N-t-Butyl-4-thia-5α-androst-1-en-3-one-17β-carboxamide.

Step 1:(N-Pyrid-3-yl)-5-hydroxy-3,5-secoandrostan-3-carboxamide-17β-(N-t-butyl)carboxamide.

From 500 mg of the product of step 5 of Example 6N-(t-butyl)-4-oxa-androstan-3-one-17β-carboxamide!, the title compoundis prepared by treatment with 1.25 equivalents of the dimethylaluminumcomplex with 3-aminopyridine by reflux in toluene for 30 min. or untilthe A-ring opening is shown to be complete by hplc or thin-layerchromatography. Purification is optional by column chromatography tofurnish the product in good yield.

Step 2:(N-Pyrid-3-yl)-5-p-toluenesulfonyloxy-3,5-secoandrostan-3-carboxamide-17.beta.-(N-t-butyl)carboxamide.

The product of step 1 in pyridine solution (50 mL) is cooled to 5° inice before 1.2 equivalents of p-toluenesulfonyl chloride is added. Afterstanding 18 hours in the refrigerator, the reaction is warmed to ambienttemperature, partitioned between water and dichloromethane and washedwith dilute bicarbonate solution to remove residual sulfonyl chloride.The organic layer is dried and evaporated to a residue, which isimmediately taken on to the iodide displacement.

Step 3:(N-Pyrid-3-yl)-5-(epi)iodo-3,5-secoandrostan-3-carboxamide-17β-(N-t-butyl)carboxamide.

The tosyl derivative of step 2 is stirred at ambient temperature with 3equivalents of dry tetrabutylammonium iodide in toluene for 30 min. andthen heated at reflux for another 2 hrs. The reaction mixture is cooled,partitioned with dichloromethane and water, and washed to removeammonium salts. After drying and solvent removal under reduced pressure,the residual amorphous material is chromatographed to provide the pureiodo compound.

Step 4:(N-Pyrid-3-yl)-5-acetylthio-3,5-secoandrostan-3-carboxamide-17β-(N-t-butyl)carboxamide.

The iodo compound of step 3 is stirred at ambient temperature with 10equivalents of thioacetic acid in toluene for 30 min. and then heated atreflux for another 2 hrs. The reaction mixture is cooled, solvents andvolatile reactants evaporated under reduced pressure, and the residualamorphous material chromatographed.

Step 5: N-t-Butyl-4-thia-5α-androstan-3-one-17β-carboxamide.

The thioacetyl derivative of step 4 is treated at ambient temperaturewith methanolic HCl (3%) until the starting material is gone bythin-layer chromatography or hplc. Product is isolated by chromatographyand recrystallized.

Step 6 N-t-Butyl-4-thia-5α-androst-1-en-3-one-17β-carboxamide.

Treatment of the product of step 5 with phenylseleninic anhydride by themethod of step 6 of Example 6 affords the product.

EXAMPLE 10 Oral Composition

As a specific embodiment of an oral composition of a compound of thisinvention, 5 mg of a compound of structural formula I of the presentinvention is formulated with sufficient finely divided lactose toprovide a total amount of 580 to 590 mg to fill a size 0 hard gelatincapsule.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe casual variations, adaptations, modifications, deletions, oradditions of procedures and protocols described herein, as come withinthe scope of the following claims and its equivalents.

What is claimed is:
 1. A compound of the structural formula below:##STR32## wherein: X is oxygen or sulfur and the 1,2 bond is optionallya double bond.
 2. The compound of claim 1 wherein X is oxygen.
 3. Amethod for treating benign prostatic hyperplasia in a human in need ofsuch treatment which comprises the administration of an effective amountof the compound of claim
 1. 4. A method for treating male-patternbaldness in a human in need of such treatment which comprises theadministration of an effective amount of the compound of claim
 1. 5. Amethod for the treatment of acne vulgaris in a human in need of suchtreatment which comprises the administration of an effective amount ofthe compound of claim
 1. 6. A method for the treatment of seborrhea in ahuman in need of such treatment which comprises the administration of aneffective amount of the compound of claim
 1. 7. A method for thetreatment of prostatic carcinoma in a human in need of such treatmentwhich comprises the administration of an effective amount of thecompound of claim
 1. 8. A pharmaceutical composition comprising thecovalent adduct is of structural formula ##STR33## and apharmaceutically acceptable carrier wherein the 1,2 bond is a doublebond.